Single-molecule detection is essential for investigating molecular interactions and dynamics but is often constrained by weak fluorescence signals and the potential for photodamage under high excitation intensities. Conventional enhancement approaches typically involve dried samples or introduce quenching and compatibility issues, making them unsuitable for in-solution measurements. This work presents a hybrid photonic platform that integrates a photonic crystal (PhC) slab with a hexagonal boron nitride (hBN) layer on top as a biocompatible substrate, offering a promising route for fluorescence enhancement under physiological conditions. Additionally, the atomically flat surface of hBN minimizes fluorophore trapping at edges or defects, enabling more uniform and reproducible single-molecule measurements across large areas.

Electromagnetic field simulations based on Rigorous Coupled-Wave Analysis (RCWA) revealed that the PhC structure can concentrate light at the hBN surface, leading to electric field intensity enhancements of up to 259-fold. To experimentally validate this, hBN flakes were successfully stamped onto the center of the PhC patches and characterized by optical microscopy and atomic force microscopy (AFM). The PhC patterns themselves were analyzed with both AFM and scanning electron microscopy (SEM). Single-stranded DNA (ssDNA) labeled with the fluorophore Atto647N was deposited and imaged by confocal fluorescence microscopy.

The experimental results demonstrated consistent fluorescence enhancement, with average increases of up to 13.15-fold across full flakes and localized enhancements up to 18.22-fold for individual fluorophores, likely positioned in high-field regions. Despite these successes, autofluorescence from the PhC introduced background signal and different flake thicknesses, complicating quantitative analysis.

This work confirms that the PhC–hBN platform can significantly enhance fluorescence in solution, providing a viable path forward for high-sensitivity single-molecule biosensing. It also highlights current challenges, such as autofluorescence and structural sensitivity, that can be subject of future research.